Mycobacterium tuberculosis has infected one third of all humans, resulting in 2-3 million deaths annually and increasing rates of coinfection with the human immunodeficiency virus (HIV). The main impediment to eradication of tuberculosis relates to the ability of M. tuberculosis to persist long term intracellularly within host tissues. Cellular immune responses, particularly activation of Th1 T cells, are crucial for killing intracellular mycobacteria and successfully resolving tuberculosis infections. Although most studies have evaluated mycobacterial protein antigen for activation of T cells, it is now known that group 1 CD1 molecules (CD1a, CD1b, CD1c) mediate T cell activation by mycobacterial glycolipids, including two classes of glycolipid antigen discovered by the applicant's group, glucose monomycolate (GMM) and mannosyl phosphodolichol (MPD). Preliminary studies indicate that CD1-restricted T cells that recognize MPD and GMM are detectable in the peripheral blood of human subjects infected with M. tuberculosis, but not naive controls, indicating that M tuberculosis infection generates glycolipid-specific T cell responses in vivo. T cells use clonally variable T cell receptors to specifically recognize several mycobacterial glycolipids without crossreactivity. T cell recognition of mycobacterial glycolipids is generally specific for the carbohydrate structure of the antigens, including a product of glycosylation reactions that is produced during intracellular growth within host tissues. Now the applicant proposes to measure polyclonal T cell responses from naive and M tuberculosis infected humans to the major classes of purified mycobacterial glycolipids typical of extracellular and intracellular growth. We will use purified CD1-presented antigens as well as mycobacterial glycolipids that are specifically upregulated during intracellular growth to measure human lymphocyte responses during the first year after infection. Antigen-specific lymphocytes will be detected using proliferation assays, antibody-capture cytokine ELISA (elispot) and glycolipid-loaded CD1 -tetramers. Glycolipid-specific T cells will be characterized with regard to restriction by CD1 -isoforms, dependence on prior infection and expression of cell surface markers of immunological memory. These studies will determine whether human infection by M tuberculosis generally results in acquired T cell responses that are specific for mycobacterial glycolipids expressed during intracellular growth. Determination of the immunodominant glycolipid targets of the human T cell response during natural tuberculosis infection will provide crucial information for development of CD1-presented glycolipids as immunomodulatory agents, including vaccines.